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The protein-tyrosine kinase ZAP-70 is implicated, together with the Src kinase p56(lck), in controlling the early steps of the T-cell antigen receptor (TCR) signaling cascade. To help elucidate further the mechanism by which ZAP-70 regulates these initial events, we used a dominant-negative mutant approach. We overexpressed in the Jurkat T-cell line ZAP-70 mutated on Tyr-492 and Tyr-493 in the putative regulatory loop of its kinase domain. This mutant inhibited TCR-induced activation of nuclear factor of activated T cells by interfering with both intracellular calcium increase and Ras-regulated activation of extracellular signal-regulated kinases. Moreover, TCR-induced phosphorylation of pp36-38, thought to play a role upstream of these pathways, was found to be reduced. In contrast, overexpression of wild-type ZAP-70 induced constitutive activation of nuclear factor of activated T cells. The ZAP-70 mutant studied here could be phosphorylated on tyrosine when associated to the TCR zeta chain and was able to bind p56(lck). This result demonstrates that Tyr-492 and Tyr-493 are not responsible for the Src homology domain 2-mediated association of p56(lck) with ZAP-70. Our data are most consistent with a model in which recruitment to the TCR allows ZAP-70 autophosphorylation and binding to p56(lck), which in turn phosphorylates Tyr-492 and/or Tyr-493 with consequent up-regulation of the ZAP-70 kinase activity. ZAP-70 will then be able to effectively control phosphorylation of its substrates and lead to gene activation.

Type

Journal article

Journal

J Biol Chem

Publication Date

20/12/1996

Volume

271

Pages

32644 - 32652

Keywords

Adaptor Proteins, Signal Transducing, Calcium, Calcium-Calmodulin-Dependent Protein Kinases, DNA-Binding Proteins, GRB2 Adaptor Protein, Humans, Lymphocyte Activation, Lymphocyte Specific Protein Tyrosine Kinase p56(lck), Macromolecular Substances, NFATC Transcription Factors, Nuclear Proteins, Phosphotyrosine, Protein-Tyrosine Kinases, Proteins, Receptors, Antigen, T-Cell, Signal Transduction, Structure-Activity Relationship, T-Lymphocytes, Transcription Factors, Tumor Cells, Cultured, ZAP-70 Protein-Tyrosine Kinase, src-Family Kinases